Nano-vesicles derived from genus morganella bacteria and use thereof

ABSTRACT

The present invention relates to vesicles derived from genus Morganella bacteria and a use thereof, the present inventors experimentally confirmed that the vesicles were significantly decreased in clinical samples derived from patients with a malignant disease such as gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer and lymphoma, a cardiovascular disease such as myocardial infarction, cardiomyopathy, atrial fibrillation, variant angina, and stroke, diabetes mellitus, and Parkinson&#39;s disease as compared to normal persons, and the vesicles suppressed the secretion of inflammatory mediators by pathogenic vesicles and suppressed the occurrence of cancer, so that the vesicles derived from genus Morganella bacteria may be usefully used for the purpose of developing a method for diagnosing a malignant disease such as gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer and lymphoma, a cardiovascular disease such as myocardial infarction, cardiomyopathy, atrial fibrillation, variant angina, and stroke, diabetes mellitus, and Parkinson&#39;s disease, and a composition for preventing or treating the diseases or an inflammatory disease

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/399,919, filed Apr. 30, 2019, which is a continuation-in-part ofPCT/KR2018/016494, filed Dec. 21, 2018, which claims the benefit ofpriority from Korean Patent Application No. 10-2018-0004603, filed Jan.12, 2018 and Korean Patent Application No. 10-2018-0158636, filed Dec.10, 2018, the contents of each of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to nano-vesicles derived from genusMorganella bacteria, and a use thereof, and more particularly, to amethod for diagnosing a malignant disease such as gastric cancer,colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer,ovarian cancer, bladder cancer, prostate cancer and lymphoma, acardiovascular disease such as myocardial infarction, cardiomyopathy,atrial fibrillation, variant angina, and stroke, diabetes mellitus, andParkinson's disease by using nano-vesicles derived from genus Morganellabacteria, and a composition for preventing or treating the disease or aninflammatory disease including the vesicles.

BACKGROUND OF THE INVENTION

Since the beginning of the 21st century, acute infectious diseasesrecognized as epidemic diseases in the past have become less important,whereas chronic diseases accompanied by immune dysfunction caused bydisharmony between humans and microbiomes have changed disease patternsas main diseases that determine the quality of life and the humanlifespan. As an intractable chronic disease in the 21st century, cancer,cardiovascular diseases, chronic lung diseases, metabolic diseases,inflammatory diseases, and neuropsychiatric diseases have become a bigproblem for public health in the country as main diseases that determinethe human lifespan and the quality of life.

Inflammation is a local or systemic protective mechanism against thedamage or infection of cells and tissues, and is typically caused by aserial biological response occurring as humoral mediators thatconstitute the immune system directly respond to the damage orinfection, or stimulate the local or systemic effector system. Examplesof a main inflammatory disease include digestive diseases such asgastritis and inflammatory enteritis, oral diseases such asperiodontitis, respiratory diseases such as asthma, chronic obstructivepulmonary disease (COPD), and rhinitis, arthritis such as degenerativearthritis and rheumatoid arthritis, and metabolic diseases such asobesity, diabetes mellitus, and hepatic sclerosis. Further, variousstudies have reported that persistent inflammation may cause cancer.

It is known that the number of microorganisms coexisting in the humanbody has reached 100 trillion, which is 10 times more than the number ofhuman cells, and the number of microorganism genes is more than 100times the number of human genes. A microbiota or microbiome refers to amicrobial community including bacteria, archaea and eukarya present in agiven habitat.

Bacteria coexisting in our body and bacteria present in the ambientenvironment secrete nanometer-sized vesicles in order to exchangeinformation on genes, low molecular compounds, proteins, and the likewith other cells. The mucosa forms a physical defense membrane throughwhich particles having a size of 200 nanometers (nm) or more cannotpass, so that bacteria coexisting in the mucosa cannot pass through themucosa, but vesicles derived from bacteria have a size of 100 nanometersor less and are absorbed into our bodies after relatively freely passingthrough epithelial cells via the mucosa.

Locally secreted bacterial-derived vesicles not only are absorbedthrough mucosal epithelial cells or skin keratinocytes to induce localinflammatory responses but also are absorbed into our bodies to bedistributed to respective organs and regulate immune and inflammatoryresponses in the organ that absorbs the vesicles. For example, vesiclesderived from pathogenic gram-negative bacteria such as Escherichia colipromote a systemic inflammatory response and blood coagulation throughvascular endothelial cell inflammatory responses when absorbed by bloodvessels, and are also absorbed into muscular cells where insulin acts tocause insulin resistance and diabetes mellitus. In contrast, vesiclesderived from beneficial bacteria may regulate diseases by regulatingimmune functions and metabolic dysfunctions by pathogenic vesicles (ChoiY W et al., Gut microbe-derived extracellular vesicles induce insulinresistance, thereby impairing glucose metabolism in skeletal muscle.Scientific Reports, 2015.).

Immune responses to factors such as vesicles derived from bacteria areTh17 immune responses characterized by the secretion of IL-17 cytokines,and IL-6 is secreted when exposed to vesicles derived from bacteria,thereby inducing Th17 immune responses. Inflammation caused by a Th17immune response is characterized by infiltration of neutrophils, andTNF-alpha, which is secreted from inflammatory cells such asmacrophages, plays an important role in the process by whichinflammation occurs.

Genus Morganella bacteria are anaerobic gram-negative rod-shapedbacteria and are known as bacteria coexisting in the intestines ofhumans and animals. Among these bacteria, Morganella morganii bacteriaare known as pathogenic bacteria causing infection after surgery,urinary tract infection, and the like. However, the fact that genusMorganella bacteria secrete vesicles out of the cells has not beenreported so far, and in particular, there have been no cases where thevesicles are applied to the diagnosis and treatment of cancer,cardiovascular diseases, metabolic diseases, inflammatory diseases, andneuropsychiatric diseases.

Thus, in the present invention, it was confirmed that a disease could bediagnosed by confirming that vesicles derived from genus Morganellabacteria were significantly decreased in clinical samples of patientswith cancer, cardiovascular diseases, metabolic diseases, inflammatorydiseases, and neuropsychiatric diseases as compared to normal persons.Further, as a result of isolating vesicles from Morganella morganiibacteria and analyzing the characteristics thereof, it was confirmedthat the vesicles could be used as a composition for preventing ortreating a malignant disease, a cardiovascular disease, a metabolicdisease, an inflammatory disease, and a neuropsychiatric disease.

As a result of intensive studies to solve the conventional problems asdescribed above, the present inventors confirmed that throughmetagenomic analysis, the content of vesicles derived from genusMorganella bacteria was significantly decreased in clinical samples frompatients with a malignant disease such as gastric cancer, colorectalcancer, pancreatic cancer, bile duct cancer, breast cancer, ovariancancer, bladder cancer, prostate cancer and lymphoma, a cardiovasculardisease such as myocardial infarction, cardiomyopathy, atrialfibrillation, variant angina, and stroke, diabetes mellitus, andParkinson's disease as compared to normal persons. In addition, it wasconfirmed that when macrophages were treated with vesicles isolated fromMorganella morganii bacteria belonging to genus Morganella bacteria, thesecretion of TNF-alpha caused by pathogenic vesicles was remarkablysuppressed, and the present invention based on this finding iscompleted.

Thus, an object of the present invention is to provide a method forproviding information for diagnosing gastric cancer, colorectal cancer,pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer,bladder cancer, prostate cancer, lymphoma, myocardial infarction,cardiomyopathy, atrial fibrillation, variant angina, stroke, diabetesmellitus, or Parkinson's disease.

Further, another object of the present invention is to provide acomposition for preventing or treating gastric cancer, colorectalcancer, pancreatic cancer, bile duct cancer, breast cancer, ovariancancer, bladder cancer, prostate cancer, lymphoma, myocardialinfarction, cardiomyopathy, atrial fibrillation, variant angina, stroke,diabetes mellitus, Parkinson's disease, or an inflammatory disease,including Morganella-derived vesicles as an active ingredient.

However, a technical problem to be achieved by the present invention isnot limited to the aforementioned problems, and the other problems thatare not mentioned may be clearly understood by a person skilled in theart from the following description.

SUMMARY OF THE INVENTION

To achieve the object of the present invention as described above, thepresent invention provides a method for providing information fordiagnosing gastric cancer, colorectal cancer, pancreatic cancer, bileduct cancer, breast cancer, ovarian cancer, bladder cancer, prostatecancer, lymphoma, myocardial infarction, cardiomyopathy, atrialfibrillation, variant angina, stroke, diabetes mellitus, or Parkinson'sdisease, the method including the following steps:

(a) extracting DNAs from vesicles isolated from samples of a normalperson and a subject;

(b) obtaining each PCR product by performing PCR using a primer pairconstructed based on a gene sequence present in 16S rDNA for theextracted DNAs; and

(c) determining a case where a content of vesicles derived from genusMorganella bacteria is lower than that of the normal person through aquantitative analysis of the PCR product as gastric cancer, colorectalcancer, pancreatic cancer, bile duct cancer, breast cancer, ovariancancer, bladder cancer, prostate cancer, lymphoma, myocardialinfarction, cardiomyopathy, atrial fibrillation, variant angina, stroke,diabetes mellitus, or Parkinson's disease.

In addition, the present invention provides a method for diagnosinggastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer,breast cancer, ovarian cancer, bladder cancer, prostate cancer,lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation,variant angina, stroke, diabetes mellitus, or Parkinson's disease, themethod including the following steps:

(a) extracting DNAs from vesicles isolated from samples of a normalperson and a subject;

(b) obtaining each PCR product by performing PCR using a primer pairconstructed based on a gene sequence present in 16S rDNA for theextracted DNAs; and

(c) determining a case where a content of vesicles derived from genusMorganella bacteria is lower than that of the normal person through aquantitative analysis of the PCR product as gastric cancer, colorectalcancer, pancreatic cancer, bile duct cancer, breast cancer, ovariancancer, bladder cancer, prostate cancer, lymphoma, myocardialinfarction, cardiomyopathy, atrial fibrillation, variant angina, stroke,diabetes mellitus, or Parkinson's disease.

As another exemplary embodiment of the present invention, the sample inStep (a) may be blood, urine, or stool.

As still another exemplary embodiment of the present invention, theprimer pair in Step (b) may be primers of SEQ ID Nos. 1 and 2.

Further, the present invention provides a pharmaceutical composition forpreventing or treating one or more diseases selected from the groupconsisting of gastric cancer, colorectal cancer, pancreatic cancer, bileduct cancer, breast cancer, ovarian cancer, bladder cancer, prostatecancer, lymphoma, myocardial infarction, cardiomyopathy, atrialfibrillation, variant angina, stroke, diabetes mellitus, Parkinson'sdisease, and an inflammatory disease, including vesicles derived fromgenus Morganella bacteria as an active ingredient.

In addition, the present invention provides a food composition forpreventing or alleviating one or more diseases selected from the groupconsisting of gastric cancer, colorectal cancer, pancreatic cancer, bileduct cancer, breast cancer, ovarian cancer, bladder cancer, prostatecancer, lymphoma, myocardial infarction, cardiomyopathy, atrialfibrillation, variant angina, stroke, diabetes mellitus, Parkinson'sdisease, and an inflammatory disease, including vesicles derived fromgenus Morganella bacteria as an active ingredient.

Furthermore, the present invention provides a method for preventing ortreating one or more diseases selected from the group consisting ofgastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer,breast cancer, ovarian cancer, bladder cancer, prostate cancer,lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation,variant angina, stroke, diabetes mellitus, Parkinson's disease, and aninflammatory disease, the method including a step of administering apharmaceutical composition including vesicles derived from genusMorganella bacteria as an active ingredient to a subject.

Further, the present invention provides a use of vesicles derived fromgenus Morganella bacteria for preventing or treating one or morediseases selected from the group consisting of gastric cancer,colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer,ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardialinfarction, cardiomyopathy, atrial fibrillation, variant angina, stroke,diabetes mellitus, Parkinson's disease, and an inflammatory disease.

As an exemplary embodiment of the present invention, the inflammatorydisease may be one or more selected from the group consisting of atopicdermatitis, acne, psoriasis, sinusitis, rhinitis, conjunctivitis,asthma, dermatitis, inflammatory collagen vascular disease,glomerulonephritis, encephalitis, inflammatory enteritis, chronicobstructive pulmonary disease, sepsis, septic shock, pulmonary fibrosis,undifferentiated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, a chronic inflammatory diseasecaused by viral or bacterial inflammation, colitis, ulcerative colitis,inflammatory bowel disease, arthritis, rheumatoid arthritis, reactivearthritis, osteoarthritis, scleriasis, osteoporosis, atherosclerosis,myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto'sthyroiditis, Grave's disease, leprosy, syphilis, Lyme disease,borreliosis, neuro-borreliosis, tuberculosis, sarcoidosis, lupus,chilblain lupus, tuberculosis lupus, lupus nephritis, systemic lupuserythematosus, macular degeneration, uveitis, irritable bowel syndrome,Crohn's disease, Sjogren syndrome, fibromyalgia, chronic fatiguesyndrome, chronic fatigue immune dysfunction syndrome, myalgicencephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease,and multiple sclerosis.

As another exemplary embodiment of the present invention, theinflammatory disease may be a disease mediated by IL-6 or TNF-α.

As still another exemplary embodiment of the present invention, thevesicles may have an average diameter of 10 to 200 nm.

As yet another exemplary embodiment of the present invention, thevesicles may be secreted naturally or artificially from genus Morganellabacteria.

As yet another exemplary embodiment of the present invention, thevesicles derived from genus Morganella bacteria may be secreted fromMorganella morganii.

The present inventors confirmed that intestinal bacteria are notabsorbed into the body, but vesicles derived from bacteria are absorbedinto the body through epithelial cells, systemically distributed, andexcreted from the body through the kidneys, liver, and lungs, and thatthrough a metagenomic analysis of vesicles derived from bacteria presentin the blood, urine, stool, or the like of a patient, vesicles derivedfrom genus Morganella bacteria present in the blood, urine, or stool ofpatients with gastric cancer, colorectal cancer, pancreatic cancer, bileduct cancer, breast cancer, ovarian cancer, bladder cancer, prostatecancer, lymphoma, myocardial infarction, cardiomyopathy, atrialfibrillation, variant angina, stroke, diabetes mellitus, and Parkinson'sdisease had been significantly decreased as compared to those in normalpersons.

Further, it was observed that when vesicles were isolated by culturingMorganella morganii which is one species of genus Morganella bacteria invitro and administered to inflammatory cells in vitro, the secretion ofinflammatory mediators by pathogenic vesicles was significantlysuppressed and the occurrence of cancer was suppressed in a canceranimal model, so that it is expected that vesicles derived from genusMorganella bacteria according to the present invention can be usefullyused for a method for diagnosing gastric cancer, colorectal cancer,pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer,bladder cancer, prostate cancer, lymphoma, myocardial infarction,cardiomyopathy, atrial fibrillation, variant angina, stroke, diabetesmellitus, and Parkinson's disease, and a composition for preventing ortreating the disease or inflammatory disease, such as a food or a drugagainst the disease or inflammatory disease.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a series of photographs capturing distribution patterns ofbacteria and vesicles derived from bacteria (EV) by time after thebacteria and the vesicles derived from bacteria were orally administeredto mice, and FIG. 1B is a result of evaluating the in vivo distributionpatterns of the bacteria and the vesicles by harvesting blood, kidneys,liver, and various organs at 12 hours after orally administering thebacteria and the vesicles.

FIGS. 2A to 2C are results of comparing the distributions of vesiclesderived from genus Morganella bacteria after performing a metagenomicanalysis on vesicles derived from bacteria present in the stool (2A),blood (2B), and urine (2C) of a patient with gastric cancer and a normalperson.

FIG. 3 is a result of comparing the distributions of vesicles derivedfrom genus Morganella bacteria after performing a metagenomic analysison vesicles derived from bacteria present in the urine of a patient withcolorectal cancer and a normal person.

FIG. 4 is a result of comparing the distributions of vesicles derivedfrom genus Morganella bacteria after performing a metagenomic analysison vesicles derived from bacteria present in the blood of a patient withpancreatic cancer and a normal person.

FIG. 5 is a result of comparing the distributions of vesicles derivedfrom genus Morganella bacteria after performing a metagenomic analysison vesicles derived from bacteria present in the blood of a patient withbile duct cancer and a normal person.

FIG. 6 is a result of comparing the distributions of vesicles derivedfrom genus Morganella bacteria after performing a metagenomic analysison vesicles derived from bacteria present in the urine of a patient withbreast cancer and a normal person.

FIGS. 7A and 7B are results of comparing the distributions of vesiclesderived from genus Morganella bacteria after performing a metagenomicanalysis on vesicles derived from bacteria present in the blood (7A) andurine (7B) of a patient with ovarian cancer and a normal person.

FIGS. 8A and 8B are results of comparing the distributions of vesiclesderived from genus Morganella bacteria after performing a metagenomicanalysis on vesicles derived from bacteria present in the blood (8A) andurine (8B) of a patient with bladder cancer and a normal person.

FIG. 9 is a result of comparing the distributions of vesicles derivedfrom genus Morganella bacteria after performing a metagenomic analysison vesicles derived from bacteria present in the urine of a patient withprostate cancer and a normal person.

FIG. 10 is a result of comparing the distributions of vesicles derivedfrom genus Morganella bacteria after performing a metagenomic analysison vesicles derived from bacteria present in the blood of a patient withlymphoma and a normal person.

FIG. 11 is a result of comparing the distributions of vesicles derivedfrom genus Morganella bacteria after performing a metagenomic analysison vesicles derived from bacteria present in the blood of a patient withmyocardial infarction and a normal person.

FIG. 12 is a result of comparing the distributions of vesicles derivedfrom genus Morganella bacteria after performing a metagenomic analysison vesicles derived from bacteria present in the blood of a patient withdilated cardiomyopathy and a normal person.

FIG. 13 is a result of comparing the distributions of vesicles derivedfrom genus Morganella bacteria after performing a metagenomic analysison vesicles derived from bacteria present in the blood of a patient withatrial fibrillation and a normal person.

FIG. 14 is a result of comparing the distributions of vesicles derivedfrom genus Morganella bacteria after performing a metagenomic analysison vesicles derived from bacteria present in the blood of a patient withvariant angina and a normal person.

FIG. 15 is a result of comparing the distributions of vesicles derivedfrom Morganella bacteria after performing a metagenomic analysis onvesicles derived from bacteria present in the blood of a patient withstroke and a normal person.

FIGS. 16A and 16B are results of comparing the distributions of vesiclesderived from genus Morganella bacteria after performing a metagenomicanalysis on vesicles derived from bacteria present in the blood (16A)and urine (16B) of a patient with diabetes mellitus and a normal person.

FIG. 17 is a result of comparing the distributions of vesicles derivedfrom genus Morganella bacteria after performing a metagenomic analysison vesicles derived from bacteria present in the urine of a patient withParkinson's disease and a normal person.

FIG. 18 is a result of evaluating apoptotic effects of vesicles derivedfrom Morganella morganii by treating macrophages with the vesiclesderived from Morganella morganii in order to evaluate the apoptoticeffects of vesicles derived from Morganella morganii.

FIGS. 19A and 19B are the evaluation results of effects on secretion ofinflammatory mediators, IL-6 (19A) and TNF-α (19B) by macrophage bypre-treatment of macrophage with the vesicles derived from Morganellamorganii before treating with the pathogenic Escherichia coli vesicles(E. coli EV), to evaluate the anti-inflammatory effects of the vesiclesderived from Morganella morganii.

FIG. 20 is an evaluation results of effects on the secretion of TNF-αinduced by Escherichia coli vesicles by pre-treatment of macrophage withvesicles derived from Morganella morganii (MMR101, MMR201) isolated fromdifferent persons before treating with the pathogenic Escherichia colivesicles (E. coli EV) to compare the anti-inflammatory effects ofvesicles derived from different strains of Morganella morganii (NC:negative control; PC: positive control; L. plantarum: Lactobacillusplantarum).

FIG. 21 is an evaluation results on the secretion of TNF-α bypre-treatment of macrophage with vesicles derived from Morganellamorganii (MMR101, MMR201) treated with heat or acid before treating withthe pathogenic Escherichia coli vesicles (E. coli EV) to evaluateeffects of the heat or acid treatment on anti-inflammatory effects ofvesicles derived from Morganella morganii (NC: negative control; PC:positive control; L. plantarum: Lactobacillus plantarum).

FIG. 22 is a protocol of administering vesicles derived from Morganellamorganii to mice in order to evaluate the anticancer efficacy ofvesicles derived from Morganella morganii.

FIG. 23 is a result of evaluating effects of cancer cells ontumorigenesis by administering Morganella morganii vesiclesintraperitoneally (IP) or orally (PO) in order to evaluate theanticancer efficacy of vesicles derived from Morganella morganii.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to vesicles derived from genus Morganellabacteria and a use thereof.

The present inventors confirmed that through a metagenomic analysis, thecontent of vesicles derived from genus Morganella bacteria wasremarkably reduced in samples from patients with gastric cancer,colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer,ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardialinfarction, cardiomyopathy, atrial fibrillation, variant angina, stroke,diabetes mellitus, and Parkinson's disease as compared to normalpersons, thereby completing the present invention based on this.

Thus, the present invention provides a method for providing informationfor diagnosing gastric cancer, colorectal cancer, pancreatic cancer,bile duct cancer, breast cancer, ovarian cancer, bladder cancer,prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrialfibrillation, variant angina, stroke, diabetes mellitus, or Parkinson'sdisease, the method including the following steps:

(a) extracting DNAs from vesicles isolated from samples of a normalperson and a subject;

(b) obtaining each PCR product by performing PCR using a primer pairconstructed based on a gene sequence present in 16S rDNA for theextracted DNAs; and

(c) determining a case where a content of vesicles derived from genusMorganella bacteria is lower than that of the normal person through aquantitative analysis of the PCR product as gastric cancer, colorectalcancer, pancreatic cancer, bile duct cancer, breast cancer, ovariancancer, bladder cancer, prostate cancer, lymphoma, myocardialinfarction, cardiomyopathy, atrial fibrillation, variant angina, stroke,diabetes mellitus, or Parkinson's disease.

The term “diagnosis” as used herein refers to determination of acondition of a disease of a patient over all aspects, in a broad sense.The contents of the determination are the disease entity, the etiology,the pathogenesis, the severity, the detailed aspects of a disease, thepresence and absence of complications, the prognosis, and the like. Thediagnosis in the present invention means determining whether gastriccancer, colorectal cancer, pancreatic cancer, bile duct cancer, breastcancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma,myocardial infarction, cardiomyopathy, atrial fibrillation, variantangina, stroke, diabetes mellitus, and Parkinson's disease occur, thelevel of the disease, and the like.

The term “nanovesicle” or “vesicle” as used herein refers to a structureconsisting of a nano-sized membrane secreted from various bacteria.

Vesicles derived from gram-negative bacteria or outer membrane vesicles(OMVs) have not only endotoxins (lipopolysaccharides) but also toxicproteins and bacterial DNA and RNA, and vesicles derived fromgram-positive bacteria also have peptidoglycan and lipoteichoic acidwhich are cell wall components of bacteria in addition to proteins andnucleic acids. In the present invention, nanovesicles or vesicles aresecreted naturally from genus Morganella bacteria or producedartificially, are in the form of a sphere, and have an average diameterof 10 to 200 nm.

The vesicles may be isolated from a culturing solution including genusMorganella bacteria by using one or more methods selected from the groupconsisting of centrifugation, ultra-high speed centrifugation, highpressure treatment, extrusion, sonication, cell lysis, homogenization,freezing-thawing, electroporation, mechanical decomposition, chemicaltreatment, filtration by a filter, gel filtration chromatography,free-flow electrophoresis, and capillary electrophoresis. Further, aprocess such as washing for removing impurities and concentration ofobtained vesicles may be further included.

The term “metagenome” as used herein also refers to a microbiome, andrefers to a total of genomes including all viruses, bacteria, fungi, andthe like in an isolated region such as soil and an animal's intestines,and is typically used as a concept of genomes explaining identificationof a large number of microorganisms at one time by using a sequenceanalyzer in order to analyze uncultivated microorganisms. In particular,the metagenome does not refer to a genome of one species, but refers toa kind of mixed genome as a genome of all species of one environmentalunit. The metagenome is, when one species is defined in the developmentprocess of omics biology, a term derived from the viewpoint of making acomplete species is made by various species interacting with each otheras well as one kind of functionally existing species. Technically, themetagenome is an object of a technique to identify all species in oneenvironment and investigate interactions and metabolism by analyzing allDNAs and RNAs regardless of species using a rapid sequence analysismethod.

In the present invention, the sample in Step (a) may be blood, urine, orstool, but is not limited thereto.

In the present invention, the primer pair in Step (b) may be primers ofSEQ ID Nos. 1 and 2.

As another aspect of the present invention, the present inventionprovides a pharmaceutical composition for preventing or treating one ormore diseases selected from the group consisting of gastric cancer,colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer,ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardialinfarction, cardiomyopathy, atrial fibrillation, variant angina, stroke,diabetes mellitus, Parkinson's disease, and an inflammatory disease,including vesicles derived from genus Morganella bacteria as an activeingredient.

As still another aspect of the present invention, the present inventionprovides a food composition for preventing or alleviating one or morediseases selected from the group consisting of gastric cancer,colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer,ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardialinfarction, cardiomyopathy, atrial fibrillation, variant angina, stroke,diabetes mellitus, Parkinson's disease, and an inflammatory disease,including vesicles derived from genus Morganella bacteria as an activeingredient.

In the present invention, the inflammatory disease may be one or moreselected from the group consisting of atopic dermatitis, acne,psoriasis, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis,inflammatory collagen vascular disease, glomerulonephritis,encephalitis, inflammatory enteritis, chronic obstructive pulmonarydisease, sepsis, septic shock, pulmonary fibrosis, undifferentiatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, a chronic inflammatory disease caused by viralor bacterial inflammation, colitis, ulcerative colitis, inflammatorybowel disease, arthritis, rheumatoid arthritis, reactive arthritis,osteoarthritis, scleriasis, osteoporosis, atherosclerosis, myocarditis,endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis,Grave's disease, leprosy, syphilis, Lyme disease, borreliosis,neuro-borreliosis, tuberculosis, sarcoidosis, lupus, chilblain lupus,tuberculosis lupus, lupus nephritis, systemic lupus erythematosus,macular degeneration, uveitis, irritable bowel syndrome, Crohn'sdisease, Sjogren syndrome, fibromyalgia, chronic fatigue syndrome,chronic fatigue immune dysfunction syndrome, myalgic encephalomyelitis,amyotrophic lateral sclerosis, Parkinson's disease, and multiplesclerosis, but is not limited thereto.

In the present invention, the inflammatory disease may be a diseasemediated by interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNF-α),but is not limited thereto.

The term “prevention” as used herein refers to all actions that suppresscancer, an inflammatory disease, a cardiovascular disease, a metabolicdisease, or a neuropsychiatric disease or delay the onset thereof viaadministration of the food or pharmaceutical composition according tothe present invention.

The term “treatment” as used herein refers to all actions that alleviateor beneficially change symptoms of cancer, an inflammatory disease, acardiovascular disease, a metabolic disease, or a neuropsychiatricdisease or delay the onset thereof via administration of the food orpharmaceutical composition according to the present invention.

The term “alleviation” used as used herein refers to all actions that atleast reduce a parameter associated with a condition to be treated, forexample, the degree of symptoms.

In an exemplary embodiment of the present invention, it was confirmedthat by orally administering bacteria and vesicles derived from bacteriato mice to evaluate the in vivo absorption, distribution, and excretionpatterns of the bacteria and the vesicles, the bacteria were notabsorbed through the intestinal mucosa, whereas the vesicles wereabsorbed within 5 minutes, systemically distributed, and excretedthrough the kidneys, the liver, and the like (see Example 1).

In another exemplary embodiment of the present invention, a bacterialmetagenomic analysis was performed by using vesicles isolated from theblood, urine, or stool of normal persons who were matched in age and sexwith patients with gastric cancer, colorectal cancer, pancreatic cancer,bile duct cancer, breast cancer, ovarian cancer, bladder cancer,prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrialfibrillation, variant angina, stroke, diabetes mellitus, or Parkinson'sdisease. As a result, it was confirmed that vesicles derived from genusMorganella bacteria were significantly decreased in samples of patientswith gastric cancer, colorectal cancer, pancreatic cancer, bile ductcancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer,lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation,variant angina, stroke, diabetes mellitus, or Parkinson's disease ascompared to samples of normal persons (see Examples 3 to 15).

In still another exemplary embodiment of the present invention, as aresult of evaluating the degree of apoptosis after treating Raw 264.7cells which are mouse macrophages with vesicles derived from Morganellamorganii (MMR101, MMR201, MMR202) at various concentrations (0.1, 1, 10μg/ml) in order to evaluate apoptotic effects of vesicles derived fromMorganella morganii (M. morganii EV) in inflammatory cells, it wasconfirmed that the apoptosis was not observed during the treatment withvesicles derived from Morganella morganii (MMR101, MMR201, MMR202) (seeExample 17).

In yet another exemplary embodiment of the present invention, as aresult of further studies to analyze characteristics of vesicles derivedfrom species Morganella morganii bacteria belonging to genus Morganellabacteria based on the results in the Examples, the Morganella morganiistrains were cultured and it was evaluated whether vesicles secretedfrom the strains exhibited anti-inflammatory effects, and as a result ofevaluating the secretion of inflammatory mediators by treatingmacrophages with vesicles derived from Escherichia coli which is acausative factor after treating the macrophages with vesicles derivedfrom Morganella morganii, it was confirmed that the vesicles derivedfrom Morganella morganii efficiently suppressed the secretion of IL-6and TNF-α by vesicles derived from Escherichia coli (see Example 18).

In yet another exemplary embodiment of the present invention, as aresult of evaluating effects of heat treatment or acid treatment on thesecretion of TNF-α by administering heat-treated or acid-treatedvesicles before treating macrophages with vesicles derived fromEscherichia coli in order to evaluate effects of heat treatment or acidtreatment on anti-inflammatory actions of vesicles derived fromMorganella morganii, it was confirmed that effects of suppressing thesecretion of TNF-α by vesicles derived from Morganella morganii were notchanged (see Example 19).

In yet another embodiment of the present invention, the Morganellamorganii strains were cultured and it was evaluated whether vesiclessecreted from the strains exhibited anti-cancer treatment effects. Forthis purpose, a cancer model was prepared by subcutaneously injecting acancer cell line and as a result of measuring the size of cancer tissuesfor 20 days after orally or intraperitoneally administering vesiclesderived from Morganella morganii to mice from 4 days before thetreatment of the cancer cell line, it was confirmed that when thevesicles were intraperitoneally and orally administered, the size ofcancer tissues was decreased as compared to that of a control, andparticularly, when the vesicles were orally administered, the size ofcancer tissues was remarkably decreased (see Example 20).

The pharmaceutical composition of the present invention may include apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier is typically used in formulation, and includes saline, sterilewater, Ringer's solution, buffered saline, cyclodextrin, a dextrosesolution, a maltodextrin solution, glycerol, ethanol, liposomes, and thelike, but is not limited thereto, and may further include other typicaladditives such as an antioxidant and a buffer, if necessary. Further,the composition may be formulated into an injectable formulation, suchas an aqueous solution, a suspension, and an emulsion, a pill, acapsule, a granule, or a tablet by additionally adding a diluent, adispersant, a surfactant, a binder, a lubricant, and the like. Withregard to suitable pharmaceutically acceptable carriers andformulations, the composition may be preferably formulated according toeach ingredient by using the method disclosed in the Remington'sliterature. The pharmaceutical composition of the present invention isnot particularly limited in formulation, but may be formulated into aninjection, an inhalant, an external preparation for skin, an oralingestion, or the like.

The pharmaceutical composition of the present invention may be orallyadministered or may be parenterally administered (for example,administered intravenously, subcutaneously, intradermally, intranasally,or the intratracheally) according to the target method, and theadministration dose may vary depending on the patient's condition andbody weight, severity of disease, drug form, and administration routeand period, but may be appropriately selected by those of ordinary skillin the art.

The pharmaceutical composition according to the present invention isadministered in a pharmaceutically effective amount. In the presentinvention, the pharmaceutically effective amount refers to an amountsufficient to treat diseases at a reasonable benefit/risk ratioapplicable to medical treatment, and an effective dosage level may bedetermined according to factors including types of diseases of patients,the severity of disease, the activity of drugs, sensitivity to drugs,administration time, administration route, excretion rate, treatmentperiod, and simultaneously used drugs, and factors well known in othermedical fields. The composition according to the present invention maybe administered as an individual therapeutic agent or in combinationwith other therapeutic agents, may be administered sequentially orsimultaneously with therapeutic agents in the related art, and may beadministered in a single dose or multiple doses. It is important toadminister the composition in a minimum amount that can obtain themaximum effect without any side effects, in consideration of all theaforementioned factors, and this may be easily determined by those ofordinary skill in the art.

Specifically, the effective amount of the pharmaceutical compositionaccording to the present invention may vary depending on the patient'sage, sex, and body weight, and generally, 0.001 to 150 mg of thecomposition and preferably, 0.01 to 100 mg of the composition, per 1 kgof body weight, may be administered daily or every other day or may beadministered once to three times a day. However, since the effectiveamount may be increased or decreased depending on the administrationroute, the severity of obesity, the gender, the body weight, the age,and the like, the administration dose is not intended to limit the scopeof the present invention in any way.

The food composition of the present invention includes a healthfunctional food composition. The food composition according to thepresent invention may be used by adding an active ingredient as is tofood or may be used together with other foods or food ingredients, butmay be appropriately used according to a typical method. The mixedamount of the active ingredient may be suitably determined depending onthe purpose of use thereof (for prevention or alleviation). In general,when a food or beverage is prepared, the composition of the presentinvention is added in an amount of 15 wt % or less, preferably 10 wt %or less based on the raw materials.

However, for long-term intake for the purpose of health and hygiene orfor the purpose of health control, the amount may be less than theabove-mentioned range.

Other ingredients are not particularly limited, except that the foodcomposition of the present invention contains the active ingredient asan essential ingredient at the indicated ratio, and the food compositionof the present invention may contain various flavorants, naturalcarbohydrates, and the like, like a typical beverage, as an additionalingredient. Examples of the above-described natural carbohydrate includecommon sugars such as monosaccharides, for example, glucose, fructoseand the like; disaccharides, for example, maltose, sucrose and the like;and polysaccharides, for example, dextrin, cyclodextrin and the like,and sugar alcohols such as xylitol, sorbitol, and erythritol. As theflavorant other than those described above, a natural flavorant(thaumatin, stevia extract (for example, rebaudioside A, glycyrrhizinand the like), and a synthetic flavorant (saccharin, aspartame and thelike) may be advantageously used. The proportion of the naturalcarbohydrate may be appropriately determined by the choice of those ofordinary skill in the art.

The food composition of the present invention may contain variousnutrients, vitamins, minerals (electrolytes), flavoring agents such assynthetic flavoring agents and natural flavoring agents, colorants andfillers (cheese, chocolate, and the like), pectic acid and saltsthereof, alginic acid and salts thereof, organic acids, protectivecolloid thickeners, pH adjusting agents, stabilizers, preservatives,glycerin, alcohols, carbonating agents used in a carbonated beverage, orthe like, in addition to the additives. These ingredients may be usedeither alone or in combinations thereof. The ratio of these additivesmay also be appropriately selected by those of ordinary skill in theart.

EXAMPLES

Hereinafter, preferred Examples for helping the understanding of thepresent invention will be suggested. However, the following Examples areprovided only to more easily understand the present invention, and thecontents of the present invention are not limited by the followingExamples.

Example 1. Analysis of In Vivo Absorption, Distribution, and ExcretionPatterns of Intestinal Bacteria and Vesicles Derived from Bacteria

In order to evaluate whether intestinal bacteria and vesicles derivedfrom bacteria were systemically absorbed through the gastrointestinaltract, an experiment was performed with the following method. A dose of50 μg of each of intestinal bacteria and vesicles derived fromintestinal bacteria labeled with fluorescence in the stomach of a mousewere administered to the gastrointestinal tract, and fluorescence wasmeasured after 0 minute, 5 minutes, 3 hours, 6 hours, and 12 hours. As aresult of observing the entire image of the mouse, as illustrated inFIG. 1A, the bacteria were not systemically absorbed through theintestinal mucosa, but the vesicles derived from bacteria weresystemically absorbed 5 minutes after administration, and fluorescencewas strongly observed in the bladder 30 minutes after administration, sothat it could be seen that the vesicles were excreted to the urinarytract. Further, it could be seen that the vesicles were present in thebody until 12 hours after administration (see FIG. 1A).

In order to evaluate the pattern in which the intestinal bacteria andthe vesicles derived from the intestinal bacteria infiltrated intovarious organs after they were systemically absorbed, 50 μg of bacteriaand vesicles derived from bacteria labeled with fluorescence wereadministered in the same manner as described above, and then the blood,heart, lungs, liver, kidneys, spleen, fat, and muscle were collected 12hours after administration. As a result of observing fluorescence in thecollected tissues, as illustrated in FIG. 1B, it could be seen that thevesicles derived from bacteria were distributed in the blood, heart,lungs, liver, kidneys, spleen, fat, and muscle, but the bacteria werenot absorbed (see FIG. 1B).

Example 2. Metagenomic Analysis of Vesicles Derived from Bacteria inClinical Sample

A clinical sample such as blood, urine, or stool was first put into a10-ml tube, suspended matter was allowed to settle down bycentrifugation (3,500×g, 10 min, 4° C.), and only the supernatant wastransferred to a new 10-ml tube. After bacteria and impurities wereremoved by using a 0.22-μm filter, they were transferred to a Centripreptube (centrifugal filters 50 kD) and centrifuged at 1,500×g and 4° C.for 15 minutes, materials smaller than 50 kD were discarded, and theresidue was concentrated to 10 ml. After bacteria and impurities wereremoved once again by using a 0.22-μm filter, the supernatant wasdiscarded by using a ultra-high speed centrifugation at 150,000×g and 4°C. for 3 hours with a Type 90Ti rotor, and an aggregated pellet wasdissolved in physiological saline (PBS).

Internal DNA was extracted out of the lipid by boiling 100 μl of thevesicles isolated by the above method at 100° C., and then cooled on icefor 5 minutes. And then, in order to remove the remaining suspendedmatter, the DNA was centrifuged at 10,000×g and 4° C. for 30 minutes,and only the supernatant was collected. And, the amount of DNA wasquantified by using Nanodrop. Thereafter, in order to confirm whetherthe DNA derived from bacteria was present in the extracted DNA, PCR wasperformed with 16s rDNA primers shown in the following Table 1 and itwas confirmed that genes derived from bacteria were present in theextracted genes.

TABLE 1 SEQ ID primer Sequence No. 16S 16S_V3_F 5′-TCGTCGGCAGCGTCAG 1rDNA ATGTGTATAAGAGACAGCC TACGGGNGGCWGCAG-3' 16S_V4_R5′-GTCTCGTGGGCTCGGAG 2 ATGTGTATAAGAGACAGGA CTACHVGGGTATCTAATCC

The DNA extracted by the above method was amplified using the 16S rDNAprimers, and then sequencing was performed (Illumina MiSeq sequencer),the results were output as a standard flowgram format (SFF) file, theSFF file was converted into a sequence file (.fasta) and a nucleotidequality score file using GS FLX software (v2.9), and then thereliability estimation for the reads was confirmed, and a portion inwhich the window (20 bps) average base call accuracy was less than 99%(Phred score<20) was removed. For the operational taxonomy unit (OTU)analysis, clustering was performed according to sequence similarity byusing UCLUST and USEARCH, the genus, family, order, class, and phylumwere clustered based on 94%, 90%, 85%, 80%, and 75% sequence similarity,respectively, classification was performed at the phylum, class, order,family, and genus levels of each OUT, and bacteria having a sequencesimilarity of 97% or more at the genus level were profiled by using the16S RNA sequence database (108,453 sequences) of BLASTN and GreenGenes(QIIME).

Example 3. Metagenomic Analysis of Vesicles Derived from Stool, Blood,and Urine Bacteria of Patient with Gastric Cancer

After a metagenomic analysis was performed on the stool from 63 patientswith gastric cancer and 126 normal persons who were matched in age andsex by extracting genes from vesicles present in the stool by the methodin Example 2, the distribution of vesicles derived from genus Morganellabacteria was evaluated. As a result, it was confirmed that vesiclesderived from genus Morganella bacteria were significantly decreased inthe stool from the patients with gastric cancer as compared to the stoolfrom the normal persons (see Table 2 and FIG. 2A).

TABLE 2 Stool Control Gastric cancer t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0001 0.0007 0.0000 0.0002 0.0472 0.20

After a metagenomic analysis was performed on the blood from 66 patientswith gastric cancer and 198 normal persons who were matched in age andsex by extracting genes from vesicles present in the blood by the methodin Example 2, the distribution of vesicles derived from genus Morganellabacteria was evaluated. As a result, it was confirmed that vesiclesderived from genus Morganella bacteria were significantly decreased inthe blood from the patients with gastric cancer as compared to the bloodfrom the normal persons (see Table 3 and FIG. 2B).

TABLE 3 Blood Control Gastric cancer t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0016 0.0096 0.0001 0.0003 0.0247 0.05

Further, after a metagenomic analysis was performed on the urine from 61patients with gastric cancer and 120 normal persons who were matched inage and sex by extracting genes from vesicles present in the urine bythe method in Example 2, the distribution of vesicles derived from genusMorganella bacteria was evaluated. As a result, it was confirmed thatvesicles derived from genus Morganella bacteria were significantlydecreased in the urine from the patients with gastric cancer as comparedto the urine from the normal persons (see Table 4 and FIG. 2C).

TABLE 4 Urine Control Gastric cancer t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0082 0.0217 0.0000 0.0001 0.0004 0.00

Example 4. Metagenomic Analysis of Vesicles Derived from Urine Bacteriaof Patient with Colorectal Cancer

After a metagenomic analysis was performed on the urine from 38 patientswith colorectal cancer and 38 normal persons who were matched in age andsex by extracting genes from vesicles present in the urine by the methodin Example 2, the distribution of vesicles derived from genus Morganellabacteria was evaluated. As a result, it was confirmed that vesiclesderived from genus Morganella bacteria were significantly decreased inthe urine from the patients with colorectal cancer as compared to theurine from the normal persons (see Table 5 and FIG. 3).

TABLE 5 Urine Control Colorectal cancer t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0211 0.0295 0.0013 0.0060 0.0002 0.06

Example 5. Metagenomic Analysis of Vesicles Derived from Blood Bacteriaof Patient with Pancreatic Cancer

After a metagenomic analysis was performed on blood from 176 patientswith pancreatic cancer and 271 normal persons who were matched in ageand sex by extracting genes from vesicles present in the blood by themethod in Example 2, the distribution of vesicles derived from genusMorganella bacteria was evaluated. As a result, it was confirmed thatvesicles derived from genus Morganella bacteria were significantlydecreased in the blood from the patients with pancreatic cancer ascompared to the blood from the normal persons (see Table 6 and FIG. 4).

TABLE 6 Blood Control Pancreatic cancer t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0020 0.0088 0.0000 0.0001 0.0003 0.00

Example 6. Metagenomic Analysis of Vesicles Derived from Blood Bacteriaof Patient with Bile Duct Cancer

After a metagenomic analysis was performed on blood from 79 patientswith bile duct cancer and 259 normal persons who were matched in age andsex by extracting genes from vesicles present in the blood by the methodin Example 2, the distribution of vesicles derived from genus Morganellabacteria was evaluated. As a result, it was confirmed that vesiclesderived from genus Morganella bacteria were significantly decreased inthe blood from the patients with bile duct cancer as compared to theblood from the normal persons (see Table 7 and FIG. 5).

TABLE 7 Blood Control Bile duct cancer t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0021 0.0091 0.0000 0.0000 0.0442 0.00

Example 7. Metagenomic Analysis of Vesicles Derived from Urine Bacteriaof Patient with Breast Cancer

After a metagenomic analysis was performed on urine from 127 patientswith breast cancer and 220 normal persons who were matched in age andsex by extracting genes from vesicles present in the urine by the methodin Example 2, the distribution of vesicles derived from genus Morganellabacteria was evaluated. As a result, it was confirmed that vesiclesderived from genus Morganella bacteria were significantly decreased inthe urine from the patients with breast cancer as compared to the urinefrom the normal persons (see Table 8 and FIG. 6).

TABLE 8 Urine Control Breast cancer t-test Taxon Mean SD Mean SD p-valueRatio g_Morganella 0.0090 0.0238 0.0002 0.0007 0.0000 0.02

Example 8. Metagenomic Analysis of Vesicles Derived from Blood and UrineBacteria of Patient with Ovarian Cancer

After a metagenomic analysis was performed on blood from 137 patientswith ovarian cancer and 139 normal persons who were matched in age andsex by extracting genes from vesicles present in the blood by the methodin Example 2, the distribution of vesicles derived from genus Morganellabacteria was evaluated. As a result, it was confirmed that vesiclesderived from genus Morganella bacteria were significantly decreased inthe blood from the patients with ovarian cancer as compared to the bloodfrom the normal persons (see Table 9 and FIG. 7A).

TABLE 9 Blood Control Ovarian cancer t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0012 0.0032 0.0000 0.0002 0.0000 0.02

Further, after a metagenomic analysis was performed on urine from 136patients with ovarian cancer and 136 normal persons who were matched inage and sex by extracting genes from vesicles present in the urine bythe method in Example 2, the distribution of vesicles derived from genusMorganella bacteria was evaluated. As a result, it was confirmed thatvesicles derived from genus Morganella bacteria were significantlydecreased in the urine from the patients with ovarian cancer as comparedto the urine from the normal persons (see Table 10 and FIG. 7B).

TABLE 10 Urine Control Ovarian cancer t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0091 0.0243 0.0000 0.0002 0.0000 0.00

Example 9. Metagenomic Analysis of Vesicles Derived from Blood and UrineBacteria of Patient with Bladder Cancer

After a metagenomic analysis was performed on blood from 96 patientswith bladder cancer and 184 normal persons who were matched in age andsex by extracting genes from vesicles present in the blood by the methodin Example 2, the distribution of vesicles derived from genus Morganellabacteria was evaluated. As a result, it was confirmed that vesiclesderived from genus Morganella bacteria were significantly decreased inthe blood from the patients with bladder cancer as compared to the bloodfrom the normal persons (see Table 11 and FIG. 8A).

TABLE 11 Blood Control Bladder cancer t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0027 0.0128 0.0000 0.0000 0.0488 0.00

Further, after a metagenomic analysis was performed on urine from 95patients with bladder cancer and 157 normal persons who were matched inage and sex by extracting genes from vesicles present in the urine bythe method in Example 2, the distribution of vesicles derived from genusMorganella bacteria was evaluated. As a result, it was confirmed thatvesicles derived from genus Morganella bacteria were significantlydecreased in the urine from the patients with bladder cancer as comparedto the urine from the normal persons (see Table 12 and FIG. 8B).

TABLE 12 Urine Control Bladder cancer t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0079 0.0224 0.0003 0.0012 0.0009 0.04

Example 10. Metagenomic Analysis of Vesicles Derived from Urine Bacteriaof Patient with Prostate Cancer

After a metagenomic analysis was performed on urine from 53 patientswith breast cancer and 159 normal persons who were matched in age andsex by extracting genes from vesicles present in the urine by the methodin Example 2, the distribution of vesicles derived from genus Morganellabacteria was evaluated. As a result, it was confirmed that vesiclesderived from genus Morganella bacteria were significantly decreased inthe urine from the patients with prostate cancer as compared to theurine from the normal persons (see Table 13 and FIG. 9).

TABLE 13 Urine Control Prostate cancer t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0042 0.0169 0.0004 0.0010 0.0050 0.09

Example 11. Metagenomic Analysis of Vesicles Derived from Blood Bacteriaof Patient with Lymphoma

After a metagenomic analysis was performed on blood from 63 patientswith lymphoma and 53 normal persons who were matched in age and sex byextracting genes from vesicles present in the blood by the method inExample 2, the distribution of vesicles derived from genus Morganellabacteria was evaluated. As a result, it was confirmed that vesiclesderived from genus Morganella bacteria were significantly decreased inthe blood from the patients with lymphoma as compared to the blood fromthe normal persons (see Table 14 and FIG. 10).

TABLE 14 Blood Control Lymphoma t-test Taxon Mean SD Mean SD p-valueRatio g_Morganella 0.0005 0.0030 0.0000 0.0000 0.1967 0.00

Example 12. Metagenomic Analysis of Vesicles Derived from Blood Bacteriaof Patient with Heart Disease

After a metagenomic analysis was performed on blood from 57 patientswith myocardial infarction and 163 normal persons who were matched inage and sex by extracting genes from vesicles present in the blood bythe method in Example 2, the distribution of vesicles derived from genusMorganella bacteria was evaluated. As a result, it was confirmed thatvesicles derived from genus Morganella bacteria were significantlydecreased in the blood from the patients with myocardial infarction ascompared to the blood from the normal persons (see Table 15 and FIG.11).

TABLE 15 Myocardial Blood Control infarction t-test Taxon Mean SD MeanSD p-value Ratio g_Morganella 0.0022 0.0103 0.0004 0.0022 0.0400 0.19

Further, after a metagenomic analysis was performed on blood from 72patients with dilated cardiomyopathy and 163 normal persons who werematched in age and sex by extracting genes from vesicles present in theblood by the method in Example 2, the distribution of vesicles derivedfrom genus Morganella bacteria was evaluated. As a result, it wasconfirmed that vesicles derived from genus Morganella bacteria weresignificantly decreased in the blood from the patients with dilatedcardiomyopathy as compared to the blood from the normal persons (seeTable 16 and FIG. 12).

TABLE 16 Blood Control Cardiomyopathy t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0022 0.0104 0.0002 0.0006 0.0133 0.08

Further, after a metagenomic analysis was performed on blood from 32patients with atrial fibrillation and 32 normal persons who were matchedin age and sex by extracting genes from vesicles present in the blood bythe method in Example 2, the distribution of vesicles derived from genusMorganella bacteria was evaluated. As a result, it was confirmed thatvesicles derived from genus Morganella bacteria were significantlydecreased in the blood from the patients with atrial fibrillation ascompared to the blood from the normal persons (see Table 17 and FIG.13).

TABLE 17 Blood Control Atrial fibrillation t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0002 0.0010 0.0000 0.0000 0.1774 0.00

Further, after a metagenomic analysis was performed on blood from 80patients with variant angina and 80 normal persons who were matched inage and sex by extracting genes from vesicles present in the blood bythe method in Example 2, the distribution of vesicles derived from genusMorganella bacteria was evaluated. As a result, it was confirmed thatvesicles derived from genus Morganella bacteria were significantlydecreased in the blood from the patients with variant angina as comparedto the blood from the normal persons (see Table 18 and FIG. 14).

TABLE 18 Blood Control Variant angina t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0028 0.0146 0.0005 0.0020 0.1736 0.18

Example 13. Metagenomic Analysis of Vesicles Derived from Blood Bacteriaof Patient with Stroke

After a metagenomic analysis was performed on blood from 115 patientswith stroke and 109 normal persons who were matched in age and sex byextracting genes from vesicles present in the blood by the method inExample 2, the distribution of vesicles derived from genus Morganellabacteria was evaluated. As a result, it was confirmed that vesiclesderived from genus Morganella bacteria were significantly decreased inthe blood from the patients with stroke as compared to the blood fromthe normal persons (see Table 19 and FIG. 15).

TABLE 19 Blood Control Stroke t-test Taxon Mean SD Mean SD p-value Ratiog_Morganella 0.0006 0.0028 0.0001 0.0014 0.1331 0.22

Example 14. Metagenomic Analysis of Vesicles Derived from Blood andUrine Bacteria of Patient with Diabetes Mellitus

After a metagenomic analysis was performed on blood from 73 patientswith diabetes mellitus and 146 normal persons who were matched in ageand sex by extracting genes from vesicles present in the blood by themethod in Example 2, the distribution of vesicles derived from genusMorganella bacteria was evaluated. As a result, it was confirmed thatvesicles derived from genus Morganella bacteria were significantlydecreased in the blood from the patients with diabetes mellitus ascompared to the blood from the normal persons (see Table 20 and FIG.16A).

TABLE 20 Blood Control Diabetes mellitus t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0054 0.0177 0.0003 0.0003 0.0012 0.05

Further, after a metagenomic analysis was performed on urine from 60patients with diabetes mellitus and 134 normal persons who were matchedin age and sex by extracting genes from vesicles present in the urine bythe method in Example 2, the distribution of vesicles derived from genusMorganella bacteria was evaluated. As a result, it was confirmed thatvesicles derived from genus Morganella bacteria were significantlydecreased in the urine from the patients with diabetes mellitus ascompared to the urine from the normal persons (see Table 21 and FIG.16B).

TABLE 21 Urine Control Diabetes mellitus t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0025 0.0119 0.0000 0.0000 0.0245 0.00

Example 15. Metagenomic Analysis of Vesicles Derived from Urine Bacteriaof Patient with Parkinson's Disease

After a metagenomic analysis was performed on urine from 39 patientswith Parkinson's disease and 79 normal persons who were matched in ageand sex by extracting genes from vesicles present in the urine by themethod in Example 2, the distribution of vesicles derived from genusMorganella bacteria was evaluated. As a result, it was confirmed thatvesicles derived from genus Morganella bacteria were significantlydecreased in the urine from the patients with Parkinson's disease ascompared to the urine from the normal persons (see Table 22 and FIG.17).

TABLE 22 Urine Control Parkinson's disease t-test Taxon Mean SD Mean SDp-value Ratio g_Morganella 0.0066 0.0182 0.0003 0.0008 0.0037 0.05

Example 16. Isolation of Vesicles from Morganella morganii CulturingSolution

Based on the Examples, after one standard strain (MMR101) from theKorean Culture Center of Microorganisms (KCCM) and two isolated strains(MMR201, MMR202) isolated from humans were cultured as Morganellamorganii (M. morganii) strains, vesicles were isolated from theculturing solution and characteristics thereof were analyzed. TheMorganella morganii (M. morganii) strains were cultured in aLuria-Bertani (LB) medium in an incubator at 37° C. until the absorbance(OD 600) became 1.0 to 1.5, and then sub-cultured. Thereafter, strainswere removed by recovering the culturing solution including the strainsand centrifuging the culturing solution at 10,000 g and 4° C. for 20minutes, and the strains were filtered with a 0.22-μm filter. Thefiltered supernatant was concentrated to a volume of 50 ml or lessthrough microfiltration by using a MasterFlex pump system (Cole-Parmer,US) with a 100 kDa Pellicon 2 Cassette filter membrane (Merck Millipore,US). The concentrated supernatant was filtered once again with a 0.22-μmfilter. Thereafter, proteins were quantified by using a BCA assay, andthe following experiments were performed on the obtained vesicles.

Example 17. Apoptotic Effects of Vesicles Derived from Morganellamorganii

In order to evaluate apoptotic effects of vesicles derived fromMorganella morganii (M. morganii EV) in inflammatory cells, after Raw264.7 cells which are mouse macrophages were treated with vesiclesderived from Morganella morganii (MMR101, MMR201, MMR202) at variousconcentrations (0.1, 1, 10 μg/ml), the degree of apoptosis wasevaluated. More specifically, Raw 264.7 cells aliquoted at 5×10⁴cells/well into a 48-well cell culture plate were treated with vesiclesderived from Morganella morganii (MMR101, MMR201, MMR202) at variousconcentrations, which was diluted with a DMEM serum-free medium, and thetreated vesicles were cultured for 12 hours. Thereafter, apoptosis wasmeasured by using EZ-CYTOX (Dogen, Korea). As a result, during thetreatment with vesicles derived from Morganella morganii (MMR101,MMR201, MMR202), apoptosis was not observed (see FIG. 18).

Example 18. Anti-Inflammatory Effects of Vesicles Derived fromMorganella morganii

In order to investigate the effects of vesicles derived from Organellarmorganii on the secretion of inflammatory mediators in inflammatorycells, Raw 264.7 cells, which are mouse macrophages, were treated withvesicles derived from Morganella morganii (MMR101) at variousconcentrations (0.1, 1, 10 μg/ml), and then the Raw 264.7 cells weretreated with E. coli derived proinflammatory pathogenic vesicles. Morespecifically, after Raw 264.7 cells were aliquoted at 1×10⁵ cells/wellinto a 24-well cell culture plate, the cells were cultured in a DMEMcomplete medium for 24 hours. Thereafter, the culture supernatant wascollected in a 1.5-ml tube and centrifuged at 3,000 g for 5 minutes, thesupernatant was recovered and stored at 4° C., and then an ELISAanalysis was performed. As a result, it was confirmed that pre-treatingthe macrophage with the vesicles derived from Morganella morganii, thesecretion of IL-6 and TNF-α induced by the vesicles derived fromEscherichia coli was remarkably suppressed (see FIGS. 19A and 19B). Inparticular, when the macrophages were pre-treated with the vesiclesderived from Morganella morganii, the degree of suppression of secretionof TNF-α from the macrophages was remarkably high as compared to themacrophages treated with Lactobacillus plantarum vesicles (see FIG.19B).

In order to evaluate anti-inflammatory effects of vesicles isolated fromMorganella morganii strains isolated from various samples, after mousemacrophage cell lines were pre-treated with vesicles derived fromMorganella morganii (MMR101, MMR201, MMR202) at various concentrations(0.1, 1, 10 μg/ml) for 12 hours, the cell lines were treated with 1μg/ml of vesicles derived from Escherichia coli, which are pathogenicvesicles, and then the secretion of TNF-α, which is an inflammatorycytokine, was measured by ELISA. As a result, it was confirmed that aTNF-α secretion-suppressing effect of vesicles derived from Morganellamorganii (MMR101, MMR201, MMR202) was higher than a TNF-αsecretion-suppressing effect by the pre-treatment of vesicles derivedfrom Lactobacillus plantarum which is a useful microorganism control(see FIG. 20). This means that vesicles, which Morganella morganiibacteria secrete, have anti-inflammatory effects regardless of a sourceof Morganella morganii.

Example 19. Effects of Heat or Acid Treatment on Anti-InflammatoryAction of Vesicles Derived from Morganella morganii

Through Example 18, anti-inflammatory effects of vesicles derived fromthe Morganella morganii standard strain (MMR101) and an isolation strain(MMR201) were identified, and furthermore, the stability of the vesiclesand characteristics of effective materials were specificallyinvestigated. For this purpose, anti-inflammatory effects were evaluatedby pre-treating macrophages (Raw 264.7) with vesicles derived from twostrains of Morganella morganii (MMR101, MMR201) boiled at 100° C. for 10minutes or treated with an acid. As a result, it was confirmed that eventhough the vesicles were boiled at 100° C. or treated with an acid,anti-inflammatory effects of vesicles derived from Morganella morganiiwere maintained (see FIG. 21). This means that the anti-inflammatoryaction of vesicles derived from Morganella morganii is stable againsttemperature and acids.

Example 20. Anti-Cancer Effects of Vesicles Derived from Morganellamorganii

Furthermore, anti-cancer effects of vesicles derived from Morganellamorganii were investigated based on the Examples. For this purpose, asillustrated in FIG. 22, a cancer model was prepared by intraperitoneallyinjecting or orally administering vesicles derived from isolated strainsof Morganella morganii (MMR201) to 6-week old C57BL/6 male mice, andsubcutaneously injecting a cancer cell line (CT26 cell) on day 4 afteradministration. After administration of the cancer cell line, thevesicles derived from isolated strains of Morganella morganii wereintraperitoneally injected or orally administered daily, and the sizesof cancer tissues were measured until day 24 (see FIG. 22). As a result,the sizes of cancer tissues were decreased in mice to which the vesicleswere administered through intraperitoneal injection and mice to whichthe vesicles were orally administered as compared to a group to whichphysiological saline was orally administered, which is a control, and inparticular, when the vesicles were orally administered, the sizes werefurther decreased (see FIG. 23). This means that when vesicles derivedfrom Morganella morganii are administered, the growth of cancer tissuesmay be efficiently suppressed.

The above-described description of the present invention is provided forillustrative purposes, and those of ordinary skill in the art to whichthe present invention pertains will understand that the presentinvention can be easily modified into other specific forms withoutchanging the technical spirit or essential features of the presentinvention. Therefore, it should be understood that the above-describedExamples are illustrative only in all aspects and are not restrictive.

It is expected that vesicles derived from genus Morganella bacteriaaccording to the present invention can be usefully used for a method fordiagnosing gastric cancer, colorectal cancer, pancreatic cancer, bileduct cancer, breast cancer, ovarian cancer, bladder cancer, prostatecancer, lymphoma, myocardial infarction, cardiomyopathy, atrialfibrillation, variant angina, stroke, diabetes mellitus, and Parkinson'sdisease, and a composition for prevention or treatment, such as a foodor drug, of the diseases or an inflammatory disease.

1. A method of reducing inflammation, the method comprisingadministering to a subject having a disorder mediated by TNF-α or IL-6 acomposition comprising an effective amount of vesicles derived frombacteria belonging to Morganella morganii.
 2. The method of claim 1,wherein the composition is a pharmaceutical composition or a foodcomposition.
 3. The method of claim 1, wherein the vesicles have anaverage diameter of 10 to 200 nm.
 4. The method of claim 1, wherein thevesicles are naturally or artificially secreted from the bacteriabelonging to Morganella morganii.
 5. The method of claim 1, wherein thecomposition is an inhalant composition.
 6. The method of claim 1,wherein the composition is administered orally or by injection.
 7. Themethod of claim 1, wherein the disorder is selected from the groupconsisting of multiple sclerosis, arthritis, psoriasis, systemic lupuserythematosus, an inflammatory bowel disease, celiac disease, asthma,chronic obstructive pulmonary disease (COPD), scleritis, vasculitis,Behcet's disease, atherosclerosis, atopic dermatitis, emphysema,periodontitis, allergic rhinitis, and gastritis.
 8. The method of claim1, wherein the composition is administered in combination with othertherapeutic agents.
 9. The method of claim 8, wherein the compositionand other therapeutic agents are administered sequentially orsimultaneously.
 10. The method of claim 1, wherein the composition isadministered daily or every other day.
 11. A method of suppressing tumorgrowth, the method comprising administering to a subject having a tumora composition comprising an effective amount of vesicles derived frombacteria belonging to Morganella morganii.
 12. The method of claim 11,wherein the composition is a pharmaceutical composition or a foodcomposition.
 13. The method of claim 11, wherein the vesicles have anaverage diameter of 10 to 200 nm.
 14. The method of claim 11, whereinthe vesicles are naturally or artificially secreted from the bacteriabelonging to Morganella morganii.
 15. The method of claim 11, whereinthe composition is an inhalant composition.
 16. The method of claim 11,wherein the composition is administered orally or by injection.
 17. Themethod of claim 11, wherein the tumor is selected from the groupconsisting of gastric cancer, pancreatic cancer, bile duct cancer,breast cancer, ovarian cancer, bladder cancer, prostate cancer, andlymphoma.
 18. The method of claim 11, wherein the composition isadministered in combination with other therapeutic agents.
 19. Themethod of claim 18, wherein the composition and other therapeutic agentsare administered sequentially or simultaneously.
 20. The method of claim11, wherein the composition is administered daily or every other day.